Cellulose is the most abundant organic polymer on Earth and represents a vast source of renewable energy. Most of this energy is stored in the recalcitrant polysaccharide cellulose, which is difficult to hydrolyze because of the highly crystalline structure, and in hemicellulose, which presents challenges because of its structural diversity and complexity. Plant cell walls are approximately composed in pinewood of lignin (30% by weight), hemicellulose (glucomannan, 20%, arabinoxylan, 10%), and crystalline cellulose (40%), which presents a major barrier to efficient use. In terrestrial ecosystems, cellulolytic microbes help drive carbon cycling through the deconstruction of biomass into simple sugars. The deconstruction is largely accomplished through the action of combinations of secreted glycoside hydrolases (GHs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), and carbohydrate binding modules (CBMs) (Baldrian and Valaskova, 2008; Cantarel, et al., 2009; Lynd, Weimer, et al., 2002; Schuster and Schmoll, 2010). Consequently, organisms from many lignocellulose-rich environments and their enzymes are being studied for new insights into overcoming this barrier.
In order to obtain the hydrolysis of crystalline cellulose, enzymes must cleave three types of glycosidic bonds. These enzymes are endocellulases, which cleave beta-1,4 glycosidic bonds that reside within intact cellulose strands in the crystalline face, non-reducing-end exocellulases, which remove cellobiose units from the non-reducing end of cellulose strands, and reducing-end exocellulases, which remove glycosyl units from the reducing-end of a cellulose strand. The endocellulolytic reaction is essential because it creates the non-reducing and reducing ends that serve as the starting point for exocellulolytic reactions. The exocellulolytic reactions are essential because they remove glycosyl groups in a processive manner from the breakages in the cellulose strand introduced by the endocellulases, thus amplifying the single initiating reaction of the endocellulases.
Trichoderma reesei and Clostridium thermocellum are well-characterized cellulose-utilizing organisms (Merino and Cherry, 2007; Bayer et al., 2008; Wilson, 2011). T. reesei is a slow-growing eukaryote fungus that secretes enzymes containing glycoside hydrolase (GH) domains fused to carbohydrate binding domains, while C. thermocellum is a strictly anaerobic prokaryote that predominantly assembles GHs and carbohydrate-binding molecules (CBMs) into a large complex called the cellulosome. Enzymes from these free-living organisms cleave polysaccharides using general acid-base catalyzed hydrolytic reactions (Vuong and Wilson, 2010). Moreover, fungal and microbial communities associated with termites (Scharf et al., 2011) shipworms (Luyten et al., 2006), and rumen (Hess et al., 2011) contribute these types of hydrolytic enzymes to their respective anaerobic niches.
Some free-living aerobes such as Cellvibrio japonicus (Ueda 107) (DeBoy et al., 2008), Streptomyces (Schlochtermeier et al., 1992; Wilson, 1992; Forsberg et al., 2011), Thermoascus aurantiacus (Langston et al., 2011; Quinlan et al., 2011) and Serratia marcescens (Vaaje-Kolstad et al., 2010) also grow on biomass polysaccharides. Recent work with some of these organisms has identified that the structurally related fungal GH61 (Langston et al., 2011; Quinlan et al., 2011) and bacterial CBM33 (Forsberg et al., 2011) families of proteins catalyze a previously unrecognized oxidative breakage of glycosidic bonds. This reaction is thought to be an endo-cleavage, with the oxidation reaction yielding gluconate and keto-sugars instead of the typically observed reducing and non-reducing sugars obtained from hydrolytic cellulases.
Actinobacteria in the genus Streptomyces are an ecologically important group, especially in soil environments, where they are considered to be vital players in the decomposition of cellulose and other biomass polymers (Cantarel et al., 2009; Crawford et al., 1978; Goodfellow and Williams, 1983; McCarthy and Williams, 1992). Streptomyces are able to utilize a wide range of carbon sources, form spores when resources are depleted, and produce antimicrobial secondary metabolites to reduce competition (Goodfellow and Williams, 1983; Schlatter et al., 2009).
Although a large number of Streptomyces species can grow on biomass, only a small percentage (14%) have been shown to efficiently degrade crystalline cellulose (Wachinger, Bronnenmeier, et al., 1989). Furthermore, the secreted cellulolytic activities of only a few species have been biochemically characterized, and still fewer species have been examined to identify key biomass degrading enzymes (Ishaque and Kluepfel, 1980; Semedo et al., 2004). Streptomyces reticuli is one of the best-studied cellulose- and chitin-degrading soil-dwelling Streptomyces; functional analyses of several important cellulases and other hydrolytic enzymes have been reported (Wachinger, Bronnenmeier, et al., 1989; Schlochtermeier, Walter, et al., 1992; Walter and Schrempf, 1996).
Furthermore, polysaccharide monooxygenase (PMO) activity with cellulose was identified using the CBM33 protein from Streptomyces coelicolor (Forsberg, et al., 2011), which suggests Streptomyces may use both hydrolytic and oxidative enzymes to deconstruct biomass. With the tremendous amount of sequence data collected in the past few years, and despite the view that Streptomyces make important contributions to cellulose degradation in the soil, genome-wide analyses of cellulolytic Streptomyces have not been reported.
In addition to their putative roles in carbon cycling in the soil, Streptomyces may also potentiate biomass deconstruction in insects through symbiotic associations (Bignell, Anderson, et al., 1991; Pasti and Belli, 1985; Pasti, Pometto, et al., 1990; Schafer, et al., 1996). Recent work has identified cellulose degrading Streptomyces associated with the pine-boring woodwasp Sirex noctilio, including Streptomyces sp. SirexAA-E (ActE) (Adams, et al., 2011). S. noctilio is a highly destructive wood-feeding insect that is found throughout forests in Eurasia and North Africa and is spreading invasively in North America and elsewhere (Bergeron, et al., 2011). While the wasp itself does not produce cellulolytic enzymes, evidence supports the role of a symbiotic microbial community that secretes biomass-degrading enzymes to facilitate nutrient acquisition for developing larvae in the pine tree (Kukor and Martin, 1983).
The white rot fungus, Amylostereum areolatum, is the best-described member of this community, and the success of Sirex infestations is thought to arise from the insect's association with this cellulolytic fungal mutualist. However, work with pure cultures has suggested that ActE and other Sirex-associated Streptomyces are more cellulolytic than A. areolatum (Adams, et al., 2011).
Optimal activity in the CBM33 enzymes apparently requires the addition of a transition metal ion such as Cu(II), Fe(III), or Mn(II) and an external reducing agent. In the laboratory, the reducing agent can be provided by ascorbate. In natural systems, the reducing function is most likely provided by another redox active protein such as cellobiose dehydrogenase (Langston et al., 2011; Quinlan et al., 2011) or some other presently unknown protein.
Needed in the art are improved compositions and organisms for digestion of lignocellulosic materials.